hemophagocytic syndrome in acute myeloid leukemia patients

Hemophagocytic syndrome in acute myeloid leukemia patients undergoing intensive chemotherapy

by Karen Delavigne, Emilie Bérard, Sarah Bertoli, Jill Corre, Eliane Duchayne, Cécile Demur,Véronique Mansat-De Mas, Cécile Borel, Muriel Picard, Muriel Alvarez, Audrey Sarry, Françoise Huguet, and Christian Récher

Haematologica 2013 [Epub ahead of print]

Citation: Delavigne K, Bérard E, Bertoli S, Corre J, Duchayne E, Demur C, Mansat-De Mas V, Borel C, Picard M, Alvarez M, Sarry A, Huguet F, and Récher C. Hemophagocytic syndrome in acute myeloid leukemia patients undergoing intensive chemotherapy. Haematologica. 2013; 98:xxxdoi:10.3324/haematol.2013.097394

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Copyright 2013 Ferrata Storti Foundation.Published Ahead of Print on October 18, 2013, as doi:10.3324/haematol.2013.097394.

1

Hemophagocytic syndrome in acute myeloid leukemia patients undergoing

intensive chemotherapy

Running title: Hyperinflammation in AML

Karen Delavigne,1,6 Emilie Bérard,2,3 Sarah Bertoli,1 Jill Corre,4 Eliane Duchayne,4

Cécile Demur,4 Véronique Mansat-De Mas,4,6 Cécile Borel,1,6 Muriel Picard,1

Muriel Alvarez,5 Audrey Sarry,1 Françoise Huguet,1 and Christian Récher1,6

1Service d’Hématologie, Centre Hospitalier Universitaire de Toulouse, Hôpital Purpan, 31059

Toulouse, France; 2Service d'Epidémiologie, Centre Hospitalier Universitaire de Toulouse,

Toulouse, F-31073, France; 3UMR 1027, INSERM-Université de Toulouse III, Toulouse, F-

31062, France; 4Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Toulouse,

Hôpital Purpan, 31059 Toulouse, France; 5Service des Maladies Infectieuses et Tropicales,

Centre Hospitalier Universitaire de Toulouse, Hôpital Purpan, 31059 Toulouse, France;

and 6Université Toulouse III Paul Sabatier, Toulouse, France.

2

Correspondence

Christian Récher, Service d’Hématologie, CHU de Toulouse, Hôpital Purpan, place du Dr

Baylac, 31059 Toulouse cedex 9, France. Email : [email protected]

Key words: hemophagocytic lymphohistiocytosis, infection-associated hemophagocytic

syndrome, malignancy-associated hemophagocytic syndrome, macrophage activation

syndrome, ferritin, acute myeloid leukemia.

Acknowledgments

The authors would like to thank the GAEL Association (Gael Adolescent Espoir Leucémie)

for their support to patients. We also thank Sarah Scotland for the corrections of the article.

3

Abstract

Hemophagocytic lymphohistiocytosis is an immune dysregulation characterized by severe

organ damages induced by a hyperinflammatory response and uncontrolled T-cell and

macrophage activation. Secondary hemophagocytic lymphohistiocytosis typically occurs in

association with severe infections or malignancies. Acute myeloid leukemia patients may be

prone to develop hemophagocytic lymphohistiocytosis because of an impaired immune

response and a high susceptibility to severe infections. In a series of 343 patients treated by

intensive chemotherapy over a 5-year period in our centre, we identified 32 patients (9.3%)

with fevers, very high ferritin levels, and marrow hemophagocytosis (hemophagocytic

lymphohistiocytosis patients). Patients with hemophagocytic lymphohistiocytosis exhibited

hepatomegaly, pulmonary or neurological symptoms, liver abnormalities, lower platelet count

and higher levels of C-reactive protein as well as prolonged pancytopenia as compared to

patients without hemophagocytic lymphohistiocytosis. A microbial etiology was documented

in 24 patients: 14 bacterial infections, 9 Herpesviridae infections and 11 fungal infections.

Hemophagocytic lymphohistiocytosis treatment consisted of corticosteroids and/or

intravenous immunoglobulin along with adapted antimicrobial therapy. Patients with

hemophagocytic lymphohistiocytosis had a median overall survival of 14.9 months,

significantly lower than patients without hemophagocytic lymphohistiocytosis (22.1 months)

(p=0.0016). Hemophagocytic lymphohistiocytosis was significantly associated with a higher

rate of induction failures mainly due to deaths in aplasia. Hemophagocytic

lymphohistiocytosis can be diagnosed in up to 10% of acute myeloid leukemia patients

undergoing intensive chemotherapy and is associated with early mortality. Fever, very high

ferritin levels and marrow hemophagocytosis represent the cornerstone of diagnosis. Further

biological studies are needed to better characterize and recognize this syndrome in acute

myeloid leukemia patients.

4

Introduction

Hemophagocytic lymphohistiocytosis (HLH) is a dysregulation of the immune system

characterized by both severe inflammation and uncontrolled activation of T-cells and

macrophages inducing severe, sometimes fatal, organ damage observed in the bone marrow,

liver, and central nervous system.(1) Patients with HLH have rapid deterioration of their

general condition, fevers, hepatosplenomegaly, profound pancytopenia, disseminated

intravascular coagulation, very high ferritin levels and hemophagocytosis in the bone marrow,

spleen or lymph nodes. Polyadenopathy, jaundice, rash, and neurological symptoms are less

frequent. This clinical syndrome can be encountered in association with various underlying

diseases, including inherited genetic defects (primary or familial HLH), malignancies, severe

infections and autoimmune disorders (secondary HLH). Primary HLH often occurs in young

children with inherited genetic defects in genes of the perforin cytotoxic pathway such as

PRF1, UNC13D, Munc 18-2, Rab27a, STX11, SH2D1A or BIRC4. Secondary HLH typically

occurs in association with severe infections (i.e, infection-associated hemophagocytic

syndrome), malignancies (i.e, malignancy-associated hemophagocytic syndrome) or

autoimmune disorders (i.e, macrophage activation syndrome). Diagnosis criteria have evolved

over time and now combine both clinical and biological features including impaired NK-cell

activity.(2-4) However, while primary HLH has been well characterized in light of the recent

discovery of mutations in several genes involved in T-cell cytotoxic activity, it is often

challenging to distinguish between true secondary HLH in adults and severe inflammatory

conditions, in which features of hemophagocytosis are encountered (i.e, critically ill

patients).(5-7) Moreover, hypomorphic mutations in PRF1, UNC13D and STBXBP2 have

recently been discovered in sporadic cases of HLH in adults.(8) Thus, there may be an overlap

between HLH and severe inflammation in some clinical contexts. An emerging concept

suggests that HLH could be a unique syndrome associated with a continuum of underlying

5

genetic risk factors and triggered by an immune challenge of varying intensity determined by

the genetic background of patients.(6)

In hematological malignancies, HLH is classically associated with specific entities, such as T-

cell or NK/T-cell lymphoma and intravascular large B-cell lymphoma, or induced by

treatment-related bacterial, viral or fungal infections and thus, frequently described as

malignancy- or infection-associated hemophagocytic syndrome.(9-11) In patients with acute

myeloid leukemia (AML), HLH has been occasionally described in case-reports. AML

patients may be prone to develop HLH due to their disease- and/or treatment-related impaired

immune response and their high susceptibility to severe infections, which act as triggering

factors.(12) Warned by several cases of HLH in our department, we sought to determine the

frequency and patterns of HLH presentation as well as its impact on prognosis in a series of

consecutive AML patients treated by intensive chemotherapy.

6

Methods

Patients

Between Jan 1, 2006, and Dec 31, 2010, all consecutive patients with a new diagnosis of

AML (except acute promyelocytic leukemia) admitted in our centre and eligible for intensive

chemotherapy were registered for this study. Diagnosis workup and treatment modalities have

been described elsewhere.(13-14) All patients had central venous catheter, bacterial digestive

tract decontamination, antibiotherapy for febrile neutropenia (piperacilline-

tazobactam/amikacine and imipenem/vancomycin/ciprofloxacin as first and second line,

respectively) and antifungal prophylaxis with posaconazole. Clinical and biological data were

recorded by K.D, A.S, S.B and C.R. Biological data, including fibrinogen, C-reactive protein,

ferritin and triglyceride levels were assessed at diagnosis and every week thereafter. Bone

marrow aspirations were routinely performed at diagnosis, day 15 of induction chemotherapy

(for patients under 60 years of age), assessment of response (~d35), in cases of unexpected

prolonged cytopenias (> d35) or in cases with suspicion of HLH (i.e, patients with

antimicrobial treatments for febrile neutropenia, and/or sudden increases in ferritinemia

and/or unexpected cytopenias). The presence of hemophagocytosis features was recorded

regardless of clinical presentation. Routine cytological analysis of May–Grünwald–Giemsa

(MGG) stained bone marrow smears was performed. Hemophagocytosis was defined by the

presence of images exhibiting macrophage-dependant phagocytosis of erythrocytes,

leukocytes, platelets and/or their precursors, regardless of the percentage of macrophages

(supplementary figure). Results of bone marrow aspirations were discussed for each patient

between cytologists and physicians during weekly meetings. This study was approved by the

Institutional Ethics Committee.

7

Study population

Three hundred and forty three patients were included. Their characteristics at diagnosis of

AML are shown in table 1. The criteria used to assign patients in the HLH group were:

hemophagocytosis in the bone marrow with unexplained fever under broad antimicrobial

treatments, and/or sudden increases in ferritin and/or unexpected non-blastic pancytopenia.

Twenty-nine patients fulfilled these criteria. Three other patients without bone marrow

hemophagocytosis exhibited strong bio-clinical evidence for the diagnosis of HLH: (i) one

patient with fever (40°C) for 12 days, hepatomegaly, cutaneous rash, rapid rise in ferritinemia

(from 1200 to 21000 µg/L) and pancytopenia with “dry tap” on bone marrow aspiration; (ii)

one patient with fever, pancytopenia, neurological symptoms, hyperferritinemia (106000

µg/L), macrophages in the cerebrospinal fluid without features of hemophagocytosis; (iii) one

patient with fever, multi-organ failure, pancytopenia and hyperferritinemia (30000 µg/L). The

total number of patients in the HLH group was 32 (HLH+ group). A group of 22 patients with

hemophagocytosis features in the bone marrow but without any clinical or biological

symptoms of HLH was also identified (HLH-/HemoPh+ group). The remaining 289 patients

had neither criterion for HLH nor bone marrow hemophagocytosis (HLH-/HemoPh- group).

Bio-clinical data were collected at diagnosis of AML for all patients and at time of HLH or

hemophagocytosis onset for HLH+ and HLH-/HemoPh+ groups. Overall, bone marrow

aspiration in HLH+ and HLH-/HemoPh+ groups were respectively performed at: (i) clinical

suspicion of HLH (n=14, 44% vs. n=0); (ii) prolonged pancytopenia (n=13, 41% vs. n=3,

14%); (iii) systematic bone marrow aspiration at diagnosis (n=1, 3% vs. n=2, 9%) or for

response evaluation (n=4, 13% vs. n=17, 77%) (p<0.0001).

Endpoints and Statistical analysis (supplementary file)

8

Results

Characteristics of patients at AML diagnosis

As shown in table 1, there were no differences in the main clinical and biological parameters

between the three groups at time of AML diagnosis. Of note, the level of ferritin was above

the upper normal limit in most patients with median ferritin levels of 725 (IQR, 362-1377),

384 (IQR, 188-1074) and 685 (IQR, 392-1594) µg/L in the HLH-/HemoPh-, HLH-/HemoPh+

and HLH+ groups, respectively. All patients received intensive chemotherapy for remission

induction, including daunorubicin + cytarabine (n=146), idarubicin + cytarabine (n=50),

idarubicin + cytarabine + lomustine (n=101), time sequential induction with daunorubicin and

cytarabine (n=13) or idarubicin + high-dose cytarabine (n=11). Other drugs, such as

gemtuzumab ozogamycin (n=19), fludarabin (n=1) or imatinib (n=2) were occasionally

added.

Characteristics of patients at HLH+ or HLH-/HemoPh+ diagnosis

Twenty-two patients developed HLH during the first induction chemotherapy, 7 during

consolidation and 3 during salvage therapy after relapse. The median time from AML

diagnosis to HLH onset was 40 days (IQR, 27-71) in the 22 patients who developed HLH

during the first induction chemotherapy. We describe the characteristics of HLH+ patients

comparatively to HLH-/HemoPh+ at HLH+ or HLH-/HemoPh+ diagnosis (table 2).

Clinically, patients in the HLH+ group had significantly more often occurrences of fever

(81%), hepatomegaly (21.9%) and respiratory symptoms (59.4%) than HLH-/HemoPh+

patients (table 2). Splenomegaly (18.8%), jaundice (25%), rash (18.8%) and neurological

symptoms (12.5%) were also frequently encountered in the HLH+ group albeit not significant

compared to the HLH-/HemoPh+ group. Biologically, HLH+ patients had significantly lower

platelets count, prothrombin time and serum albumin level as well as higher levels of C-

9

reactive protein, whereas there were no differences in serum sodium level, creatinine,

triglycerides and fibrinogen levels between the two groups. Liver abnormalities were

significantly associated with HLH as indicated by higher bilirubin levels and cholestasis

markers, including γGT and alkaline phosphatase.

Variations of ferritin levels

At time of bone marrow aspiration performed for HLH diagnosis or response assessment, the

median serum ferritin level was 5093 µg/L (IQR, 2826-11307) in HLH+ patients compared to

1866.5 µg/L (IQR, 660-2789) in HLH-/HemoPh+ patients (p=0.0004) (table 3). During the

period following the bone marrow aspiration (until response to HLH treatment or death), the

median maximal value of ferritin was 6953.5 µg/L (IQR, 4425.5-15305.5) in the HLH+ group

compared to 3373 µg/L (IQR, 1808-5149) in the HLH-/HemoPh+ patients (p=0.0071).

Moreover, 15 patients (47%) in the HLH+ group and only 2 (11%) in the HLH-/HemoPh+

group, had a ferritin level higher than 8000 µg/L (p=0.0104). We also studied the variation in

serum ferritin either in the week prior to diagnosis of HLH in 22 patients or prior to

hemophagocytosis on bone marrow aspiration in 12 HLH-/HemoPh+ patients. The median

increase in ferritin was 1421 µg/L (IQR, -287-8746) and 73.5 µg/L (IQR, -836-802.5) in

HLH+ and HLH-/HemoPh+ patients, respectively (p=0.1395). The median number of red cell

packs received by HLH+ patients in the three-month period before the diagnosis of HLH was

14 (IQR, 8-20) compared to 7 (IQR, 2-10) in HLH-/HemoPh+ patients (p=0.0048). There was

no significant correlation between the maximal level of serum ferritin and the number of red

cell packs in HLH+ patients (Spearman correlation coefficient, Rho=-0.14; p=0.46).

Etiology

A potential infectious etiology functioning as trigger for HLH has been found in 24 patients

(75%): 14 patients with bacterial infections, which were mainly septicemia and pneumonia, 9

10

patients with Herpesviridae infections and 11 patients with fungal infections, which were

mainly invasive aspergillosis (table 4). No mycobacterial infections have been documented.

During the induction phase, bacterial or fungal infections were documented in 15 HLH+

patients (46.9%) and in 91 HLH-/HemoPh- patients (31.5%) (p=0.079). Parenteral nutrition

and growth factors have been occasionally associated with hemophagocytosis.(15-16)

Parenteral nutrition was delivered in 10 HLH+ (31%) and 6 HLH-/HemoPh+ (27%) patients,

respectively (p=0.75). Before HLH and hemophagocytosis diagnosis, 20 HLH+ (62.5%) and

6 HLH-/HemoPh+ (27%) patients received G-CSF (p=0.01).

Treatment of HLH

Twenty nine patients (90.6%) received a specific treatment for HLH in a median time of 24

hours after diagnosis. First line treatment was corticosteroids (CS) in 5 patients, intravenous

immunoglobulins (IVIg) in 16 patients, CS + IVIg in 5 patients, CS + etoposide in 2 patients

and CS + IVIg + ciclosporine in one patient. Overall, CS and IgIV were used as the first line

treatment in 13 (44%) and 22 (76%) patients, respectively. The distribution of treatment

modalities according to the type of infection associated with HLH is shown in figure 1. No

patient with invasive fungal infection received CS as part of the first-line treatment. Patients

could receive second-line treatment with CS, IVIg or ciclosporine according initial response.

Although no response criteria are specifically defined for HLH, we judged that rapid fever

disappearance (within 48 hours) and resolution of cytopenias were the most relevant markers

of response. In our data, the ferritin decrease in the few days (d0 to d7 or d15) following the

treatment of HLH+ patients (n=29), did not permit to differentiate between responders and

non-responders. Of the 13 patients treated by CS, 9 (69%) had complete resolution of fever

whereas only 6 out of 16 patients (37.5%) treated by IVIg alone became apyretic.

Improvement of cytopenias was observed in 1/5 (20%) patients treated by CS alone and in

7/16 (44%) patients treated by IVIg alone. Of the 5 patients treated by CS + IVIg as the first

11

line, 4 had fever resolution and 3 had improvement of cytopenias. Of note, the response rate

was not associated with the type of underlying infection: 43% (n=6; p=0.688) in bacterial

infections, 33% (n=3; p=0.397) in viral infections and 36% (n=4; p=0.388) in fungal

infections. Overall, 20 patients (62.5%) had a resolution of symptoms after adapted antibiotic

or antifungal therapy and specific treatment of the HLH.

Outcome of HLH patients

The median overall survival was 14.9 months (IQR, 2.9-41.7) in the HLH+ group and 22.1

months (IQR, 9.2-85.6) in the HLH- group (HLH-/HemoPh- and HLH-/HemoPh+),

respectively (log-rank test, p=0.0016) (figure 2). In Cox proportional hazards models adjusted

for prognostic factors of AML, including age, secondary AML, white blood cell count,

cytogenetics, performance status and consolidation treatment by autologous or allogeneic

stem cell transplantation, HLH+ patients had a significantly increased risk of death compared

to HLH- patients (Hazard Ratio, 2.05, 95% CI, 1.31 to 3.22, p=0.002) (table 5). The adjusted

risk of HLH-/HemoPh+ patients did not significantly differ compared with HLH-/HemoPh-

patients. To describe in more detail the impact of HLH onset in the context of AML patients,

we focused on the 22 patients (and 16 patients) in whom HLH (or HLH-/HemoPh+) was

diagnosed in the course of intensive chemotherapy for first remission induction. In these

patients, the duration of neutropenia (37 days, IQR 29-59) tended to be longer than in HLH-

/HemoPh+ patients (28.5 days, IQR 24-38; p=0.158). In the HLH+ group, 9/22 patients

(40.9%) reached CR or CRi compared to 214/289 (74.1%) and 15/16 (93.8%) in the HLH-

/HemoPh- and HLH-/HemoPh+ groups, respectively (p=0.001) (table 6). The lower response

rate in the HLH+ group was associated with higher rates of hypoplastic death and resistant

disease, resulting in a significantly higher mortality rate at 3 months (36.4% in the HLH+

group compared to 12.5% in the HLH-/HemoPh- and HLH-/HemoPh+ groups, p=0.011).

12

Discussion

It has been recently suggested that hyperinflammatory disorders, such as HLH, macrophage

activating syndrome, malignancy-associated hemophagocytic syndrome or systemic

inflammatory systemic response, could be linked by a common pathophysiology involving

aberrant cytokine release, the so-called cytokine storm.(6, 17) We show here that up to 10%

of AML patients undergoing intensive chemotherapy displayed a clinical presentation

consistent with HLH although the strict criteria defined for pediatric patients were not met in

our series.(4) However, it should be emphasized that there is no current consensus on the

HLH criteria for adult patients.(18) The comparison of patients without symptoms of HLH

but hemophagocytosis in the bone marrow and HLH+ patients, showed that

hemophagocytosis itself is not a specific feature of HLH. We also acknowledge that the

distinction between HLH and other febrile conditions (i.e. pure infectious complications)

could be challenging in the setting of intensive chemotherapy course. Thus, a comparison

with other febrile, neutropenic AML patients would be necessary to better assess the specific

markers of HLH in those patients. Unfortunately, we did not collect data of all AML patients

at the time of first febrile episode during the period of this study to perform such a

comparison. However, virtually all AML patients undergoing intensive chemotherapy are

neutropenic for a long period of time (usually 21 days) and it is quite exceptional that these

patients remain afebrile. In our data base including 643 patients undergoing intensive

chemotherapy between 2000 and 2010, 136 patients had bacterial and 60 fungal documented

infections (~30%).(13) In this series, bacterial or fungal infections were documented in 31.5% of

HLH-/HemoPh- patients during the induction phase, indicating that a substantial proportion of

neutropenic patients with documented infections did not develop the particular clinical picture of

HLH+ patients. Thus, we feel confident that the clinical picture we describe here in roughly

10% of patients encompass bona fide criteria to distinguish HLH patients from other

13

neutropenic AML patients. Furthermore, most of the classical criteria of HLH seem

unsuitable when applied to leukemia due to the nature of the disease. This is especially the

case for pancytopenia related to bone marrow involvement and intensive chemotherapy and

the ferritin levels that are nearly always greater than 500 µg/L at time of AML diagnosis and

further increased by repeated red blood cell transfusions. Overall, we have identified criteria

that could help to identify HLH in AML patients (table 7). These criteria could be further

improved by specific prospective studies assessing the T-cell and NK cell activity, sCD25,

genetic screen and cytokine profiling in the setting of AML patients treated by intensive

chemotherapy. Indeed, it will be interesting to determine whether those patients have a

genetic background (mild hypomorphic mutations, polymorphism or complex polygenic

traits) predisposing them to HLH during intensive chemotherapy.(17) Additionally, a large

microbiological screen including viral testing should be part of the initial workup in HLH+

patients. In this study, the onset of HLH in the course of AML therapy had a strong impact on

prognosis as shown by the multivariate analysis. We show that HLH+ patients had a higher

rate of death in aplasia, resistant disease and a shorter overall survival. As various infectious

causes have been identified, with an overrepresentation of herpesviridae and invasive

aspergillosis, these early deaths are probably due to severe infections. However, HLH induced

liver and/or pulmonary involvement and prolonged pancytopenia that undoubtedly worsened

these infections. Although not significant in our study, it is also noteworthy that HLH+

patients had a higher rate of induction chemotherapy failure suggesting that uncontrolled

disease could have also impacted survival. The fact that less patients with HLH failed to

achieve complete response could underline the role of the malignant disease as a trigger for

HLH. One could therefore argue that HLH is a surrogate marker for failure to chemotherapy.

Alternatively and somewhat provocative, inflammatory response could also improve leukemia

cell survival and subsequent resistance to chemotherapy.

14

It is therefore critical to detect early onset of HLH in AML patients. A typical presentation is

a patient with a high fever even under broad antibiotic treatments, a rapid rise in the ferritin

level (sometimes to extraordinarily high levels) and liver abnormalities. This clinical picture

should suggest bone marrow aspiration for determination of hemophagocytosis, although this

latter criterion is not an absolute prerequisite for diagnosis. A large infectious workup is also

required to maximize antimicrobial treatment since we documented an infectious agent as a

potential trigger of HLH in 75% of cases. HLH treatments, which should be started promptly,

remain challenging, particularly in aplastic patients having received intensive chemotherapy.

Corticosteroids, etoposide-based regimens, ciclosporin and antithymocyte globulin are

currently administered in pediatric patients.(18) Immunomodulation with IVIg has also been

used in virus-associated HLH.(19) Because of the profound pancytopenia induced by

chemotherapy, etoposide has been exceptionally used in our patients. In fact, most were

treated by corticosteroids (either prednisolone or dexamethasone) or IVIg as determined by

the underlying infection. Although our study does not establish a standard of care in this

situation, we believe that the combination of IVIg and corticosteroids (short pulse then taper)

could be the treatment of choice. Indeed, corticosteroids induced a rapid improvement in

symptoms and overall condition, while IVIg were associated with a better survival in HLH+

patients (data not shown). Other more rational therapies such as anti-cytokines (e.g.,

tocilizumab) should be assessed in this setting.(20)

Our study also revealed quite unexpectedly that patients with bone marrow hemophagocytosis

but without HLH did particularly well in terms of complete response and overall survival with

a median of 85.6 months (IQR, 27.9-85.6). In most of these HLH-/HemoPh+ patients, bone

marrow aspiration was performed to assess the response to chemotherapy. Although

speculative, hemophagocytosis in this setting could be a marker of innate immunity against

leukemic cells. Indeed, macrophages express signal regulatory protein alpha (SIRPα) that

15

when activated by its ligand, CD47, induces an intracellular signalization resulting in

inhibition of phagocytosis.(21) It has been demonstrated that increased CD47 expression by

leukemic stem cells inhibits macrophage activity and is associated with poor prognosis in

AML.(22-23) Conversely, patients with low CD47 expression are more chemosensitive,

suggesting that there may be a correlation between marrow hemophagocytosis and CD47

expression in AML patients.

To conclude, HLH onset can be encountered in up to 10% of AML patients undergoing

intensive chemotherapy and is associated with induction failure and early mortality. Fever, a

very high ferritin level and marrow hemophagocytosis represent the cornerstone of diagnosis,

which must be rapidly established to adapt antimicrobial treatments and to promptly introduce

immunomodulation. Further studies are needed to better characterize this syndrome in AML

patients using genetic screening, cytokine monitoring or T-cell and NK-cell functional

studies.

Authorship and disclosures

Author’s Contributions

K.D. collected and analyzed data; E.B. performed statistical analysis and wrote the paper;

S.B. collected and analyzed data; J.C.; E.D.; C.D. and V.d.M. performed cytological analysis;

C.B; M.P.; M.A and F.H.; treated patients; A.S. collected data; C.R. treated patients, collected

and analyzed data, and wrote the paper. All the authors checked the final version of the

manuscript.

Disclosure of Conflicts of Interest: the authors declare no competing financial interests.

16

References

1. Janka GE. Hemophagocytic syndromes. Blood Rev. 2007;21(5):245-53. 2. Arico M, Janka G, Fischer A, Henter JI, Blanche S, Elinder G, et al. Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society. Leukemia. 1996;10(2):197-203. 3. Henter JI, Elinder G, Ost A. Diagnostic guidelines for hemophagocytic lymphohistiocytosis. The FHL Study Group of the Histiocyte Society. Semin Oncol. 1991;18(1):29-33. 4. Henter JI, Horne A, Arico M, Egeler RM, Filipovich AH, Imashuku S, et al. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007;48(2):124-31. 5. Castillo L, Carcillo J. Secondary hemophagocytic lymphohistiocytosis and severe sepsis/ systemic inflammatory response syndrome/multiorgan dysfunction syndrome/macrophage activation syndrome share common intermediate phenotypes on a spectrum of inflammation. Pediatr Crit Care Med. 2009;10(3):387-92. 6. Risma K, Jordan MB. Hemophagocytic lymphohistiocytosis: updates and evolving concepts. Curr Opin Pediatr. 2012;24(1):9-15. 7. Strauss R, Neureiter D, Westenburger B, Wehler M, Kirchner T, Hahn EG. Multifactorial risk analysis of bone marrow histiocytic hyperplasia with hemophagocytosis in critically ill medical patients--a postmortem clinicopathologic analysis. Crit Care Med. 2004;32(6):1316-21. 8. Zhang K, Jordan MB, Marsh RA, Johnson JA, Kissell D, Meller J, et al. Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH. Blood. 2011;118(22):5794-8. 9. Falini B, Pileri S, De Solas I, Martelli MF, Mason DY, Delsol G, et al. Peripheral T-cell lymphoma associated with hemophagocytic syndrome. Blood. 1990;75(2):434-44. 10. Ferreri AJ, Dognini GP, Campo E, Willemze R, Seymour JF, Bairey O, et al. Variations in clinical presentation, frequency of hemophagocytosis and clinical behavior of intravascular lymphoma diagnosed in different geographical regions. Haematologica. 2007;92(4):486-92. 11. Han AR, Lee HR, Park BB, Hwang IG, Park S, Lee SC, et al. Lymphoma-associated hemophagocytic syndrome: clinical features and treatment outcome. Ann Hematol. 2007;86(7):493-8. 12. Bertozzi AI, Suc A, Rubie H, Duchayne E, Demur C, Robert A. [Hemophagocytic syndrome associated with neutropenia after chemotherapy]. Arch Pediatr. 2002;9(2):125-9. 13. Bertoli S, Berard E, Huguet F, Huynh A, Tavitian S, Vergez F, et al. Time from diagnosis to intensive chemotherapy initiation does not adversely impact the outcome of patients with acute myeloid leukemia. Blood. 2013;121(14):2618-26. 14. LaRochelle O, Bertoli S, Vergez F, Sarry JE, Mansat-De Mas V, Dobbelstein S, et al. Do AML patients with DNMT3A exon 23 mutations benefit from idarubicin as compared to daunorubicin? A single center experience. Oncotarget. 2011;2(11):850-61. 15. Wang S, Degar BA, Zieske A, Shafi NQ, Rose MG. Hemophagocytosis exacerbated by G-CSF/GM-CSF treatment in a patient with myelodysplasia. Am J Hematol. 2004;77(4):391-6. 16. Roth B, Grande PO, Nilsson-Ehle P, Eliasson I. Possible role of short-term parenteral nutrition with fat emulsions for development of haemophagocytosis with multiple organ failure in a patient with traumatic brain injury. Intensive Care Med. 1993;19(2):111-4. 17. Usmani GN, Woda BA, Newburger PE. Advances in understanding the pathogenesis of HLH. Br J Haematol. 2013;161(5):609-22. 18. Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis. Blood. 2011;118(15):4041-52. 19. Chen RL, Lin KH, Lin DT, Su IJ, Huang LM, Lee PI, et al. Immunomodulation treatment for childhood virus-associated haemophagocytic lymphohistiocytosis. Br J Haematol. 1995;89(2):282-90. 20. Teachey DT, Rheingold SR, Maude SL, Zugmaier G, Barrett DM, Seif AE, et al. Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy. Blood. 2013;121(26):5154-7. 21. Barclay AN, Brown MH. The SIRP family of receptors and immune regulation. Nat Rev Immunol. 2006;6(6):457-64.

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22. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr., et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286-99. 23. Jaiswal S, Jamieson CH, Pang WW, Park CY, Chao MP, Majeti R, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell. 2009;138(2):271-85.

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Table 1: Characteristics of patients at diagnosis of AML

HLH-/HemoPh-

n= 289 (84.3%)

HLH-/HemoPh+

n=22 (6.4%)

HLH+

n=32 (9.3%)

Age (years)

Median (IQR)

59 (49-66)

55.5 (41-61)

62 (46-69)

Gender

Male, n (%)

Female, n (%)

166 (57.4)

123 (42.6)

12 (54.5)

10 (45.5)

19 (59.4)

13 (40.6)

ECOG performance status

0-1, n (%)

≥2, n (%)

Unknown, n (%)

196(67.8)

38 (13.1)

55 (19)

20 (90.9)

0 (0)

2 (9.1)

21 (65.6)

4 (12.5)

7 (21.9)

AML status

De novo, n (%)

Secondary, n (%)

Unknown, n (%)

231 (79.9)

54(18.7)

4 (1.4)

18 (81.8)

4 (18.2)

0 (0)

23 (71.9)

8 (25)

1 (3.1)

Previous MDS, n (%) 27 (9.3) 1 (4.5) 5 (15.6)

Previous autoimmune disorder, n (%) 1 (0.4) 0 (0) 1 (3.1)

Extramedullary involvement

Yes, n (%)

No, n (%)

Unknown, n (%)

73 (25.3)

171 (59.2)

45 (15.6)

5 (22.7)

16(72.7)

1 (4.5)

12 (37.5)

17 (53.1)

3 (9.4)

WBC (G/L)

Median (IQR)

10.6 (3.1-42.3)

7.4 (2.2-41.3)

7.7 (2.9-20.6)

Hemoglobin (g/dL)

N

Median (IQR)

289

9.6 (8.4-11.3)

22

11.1 (7.8-12.8)

31

9.9 (9-11.1)

Platelets (G/L)

N

Median (IQR)

286

70 (38-118)

21

96 (67-134)

31

53 (16-162)

Lymphocytes (G/L)

N

277

22

31

Median (IQR) 2.1 (1.1-3.6) 1.7 (1.2-6.3) 1.8 (0.9- 2.8)

Bone marrow blasts (%)

N

Median (IQR)

278

64.5 (38-85)

21

51 (37-82)

29

44 (24-78)

Multilineage dysplasia

Yes, n (%)

No, n (%)

40 (13.8)

233 (80.6)

4 (18.2)

16 (72.7)

8 (25)

21 (65.6)

19

Unknown, n (%) 16 (5.5) 2 (9.1) 3 (9.4)

Cytogenetics

Favorable, n (%)

Intermediate, n (%)

Adverse, n (%)

Unknown, n (%)

26 (9)

198 (68.5)

62 (21 .5)

3 (1)

3 (13.6)

14 (63.6)

5 (22.7)

0 (0)

2 (6.3)

24 (75)

5 (15.6)

1 (3.1)

Ferritin (µg/L)

N

Median (IQR)

153

725 (362-1377)

15

384 (188-1074)

25

685 (392-1594)

Total percentages differ from 100% because of rounding

HLH: Hemophagocytic lymphohistiocytosis; HemoPh+: bone marrow hemophagocytosis;

MDS: Myelodysplasic syndrome; IQR: Inter-Quartile Range.

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Table 2: Comparisons of HLH+ and HLH-/HemoPh+ patients at HLH+ or HemoPh+ diagnosis

HLH+ n=32

HLH-/HemoPh+ n=22

p-value

Fever-n (%) 26 (81.3) 6 (27.3) .0001

Splenomegaly-n (%) 6 (18.8) 0 (0) .0706

Hepatomegaly-n (%) 7 (21.9) 0 (0) .0335

Icterus-n (%) 8 (25) 1 (4.5) .0666

Rash-n (%) 6 (18.8) 1 (4.5) .2197

Neurological symptoms-n (%) 4 (12.5) 0 (0) .1368

Respiratory symptoms-n (%) 19 (59.4) 2 (9.1) .0002 WBC (G/L)-Median (IQR) 0.4 (0.2- 1.6) 0.9 (0.5-4.1) .0723 Hemoglobin (g/dL)-Median (IQR) 9 (8.3-9.7) 9.7 (9- 10.8) .0628 Platelets (G/L)-Median (IQR) 18.5 (11- 28) 47.5 (12-155) .0494

PTT (%) N Median (IQR)

30

66.5 (57- 78)

20

84 (69.5-98)

.0013

Fibrinogen N Median (IQR)

26

5.1 (4.2-5.9)

18

4.4 (3.1-5.3)

.1042

Natremia (mmol/L) -Median (IQR) 135.5(133-138) 138 (134-139) .2351

Triglycerides (mmol/L) N Median (IQR)

30

1.3 (0.9- 2.5)

22

1.2 (0.8-1.8)

.3788

Creatinine (>1.5 x ULN)-n (%) 5 (15.6) 1 (4.5) .3826

Albumin (g/L) N Median (IQR)

29

27 (25- 30)

22

33 (29-37)

.0005

AST (UI/L)-Median (IQR) 31.5 (18- 103.5) 26 (19-38) .1835 ALT (UI/L)-Median (IQR) 31 (15.5-107.5) 29 (16-37) .2241 AST or ALT (>5 x ULN)-n (%) 7 (21.9) 0 (0) .0335

Alkaline phosphatase (UI/L) Median (IQR) >2 x ULN-n (%)

427 (251.5-635.5)

10 (31.3)

214 (175-276)

1 (4.5)

.0005 .0189

γGT (UI/L) Median (IQR) >5 x ULN-n (%)

213 (136-333.5)

19 (59.4)

59.5(34-119)

3 (13.6)

.0001 .0008

Bilirubin (µmol/L) N Median (IQR) >ULN-n (%)

30

13.5 (10-49) 13(40.6)

21

10 (8-11) 3 (13.6)

.0066 .0328

C-reactive protein (mg/L) N Median (IQR)

30

115.5 (57- 178)

19

16 (5- 92)

.0005

21

HLH: Hemophagocytic lymphohistiocytosis; HemoPh+: bone marrow hemophagocytosis.

WBC: White Blood Cells, ULN: Upper Limit of Normal, PTT: Prothrombin time; AST:

aspartate transaminases; ALT: alanine transaminases; γGT: gamma-glutamyltranspeptidase; N

is only mentioned when there is unknown data. Bio-clinical data were collected at time of HLH or

hemophagocytosis onset for HLH+ and HLH-/HemoPh+ groups.

22

Table 3: Ferritin levels in HLH+ and HLH-/HemoPh+ patients

HLH+

N=32

HLH-/HemoPh+

N=22 p-value

Ferritin (µg/L)

At HLH/HemoPh+ diagnosis

N

Median (IQR)

31

5093 (2826-11307)

18

1866.5 (660-2789)

0.0004

Maximal value during

evolution*

N

Median (IQR)

32

6953.5 (4425.5-15305.5)

18

3373 (1808-5149)

0.0071

> 500, n (%) 32 (100) 17 (94.4) 0.3600

> 3000, n (%) 26 (81.3) 11 (61.1) 0.1797

> 8000, n (%) 15 (46.9) 2 (11.1) 0.0104

> 10000, n (%) 13 (40.6) 2 (11.1) 0.0287

HLH: Hemophagocytic lymphohistiocytosis; HemoPh+: bone marrow hemophagocytosis. * Evolution represents the time between HLH diagnosis and response to anti-HLH treatment or death.

23

Table 4: Infections at HLH+ or HemoPh+ diagnosis.

HLH+ N=32

HLH-/HemoPh+ N=22

Bacterial Infection Site Septicemia Pneumonia Soft tissus Other Bacteria Enterobacteria Cocci gram + Stenotrophomonas maltophlia Other

14 6 4 2 2 5 3 3 3

7 2 0 4 1 0 6 0 1

Viral Infection EBV HHV6 HHV8 HSV1 CMV Influenzae Rhinovirus

9 2 4 1 1 1 0 0

4 1 0 0 1 0 1 1

Fungal Infection Invasive aspergillosis Lung (possible) Lung (probable) Sinus Mucormycosis Lung

11 2 6 2 1

0 0 0 0 0


Of note, 4 HLH+ patients had concomitant viral and bacterial infection, 3 had viral and fungal

infection and 3 other bacterial and fungal infection. Two HLH-/HemoPh+ patients had

concomitant viral and bacterial infection.

24

Table 5: Multivariate Cox proportional-hazards model for overall survival in HLH+

patients versus HLH- patients (including HLH-/HemoPh+ and HLH-/HemoPh-)

Hazard Ratio 95% Confidence Interval P value

HLH- 1.00

HLH+ 2.05 1.31-3.22 0.002

Age 1.01 1.00-1.02 0.028

De novo AML 1.00

Secondary AML 1.63 1.18-2.26 0.003

WBC ≤ 50 G/liter 1.00

WBC > 50 G/liter 1.64 1.16-2.30 0.005

Favorable cytogenetic risk 1.00

Intermediate cytogenetic risk 1.96 1.01-3.83 0.047

Adverse cytogenetic risk 3.52 1.72-7.19 0.001

WHO performance status=0 1.00

WHO performance status=1 1.32 0.93-1.87 0.116



No-SCT 1.00

Autologous-SCT 0.32 0.14-0.73 0.007

Allogeneic-SCT 0.63 0.42-0.95 0.027


WBC: White Blood Cell. SCT: Stem Cell Transplantation.

25

Table 6: Outcome of patients after induction remission chemotherapy (in patients with HLH+ or HLH-/HemoPh+ during induction remission chemotherapy)

HLH-/HemoPh- n= 289

HLH-/HemoPh+ n=16

HLH+ n=22

P-value

Response-n (%) CRi 33 (11.4) 0 (0) 5 (22.7) 0.093 CR 181 (62.6) 15 (93.8) 4 (18.2) <0.001* CR or CRi 214 (74.1) 15 (93.8) 9 (40.9) 0.001**

Induction failure-n (%) Early deaths 11 (3.8) 0 (0) 0 (0) 1 Deaths in aplasia 15 (5.2) 0 (0) 7 (31.8) <0.001*** Resistant disease 40 (13.9) 1 (6.3) 6 (27.3) 0.167

Relapse £ -n (%) 107 (48.2) 5 (33.3) 5 (62.5) 0.387

3-month mortality -n (%) 36 (12.5) 2 (12.5) 8 (36.4) 0.011****

HLH: Hemophagocytic lymphohistiocytosis; HemoPh+ : bone marrow hemophagocytosis; CRi : complete response with incomplete blood count recovery ; CR : complete response.

£ In patients with CR or CRi or complete response with two cycles of induction.

*According to Bonferroni correction p-value=0.033 for HLH-/HemoPh- vs HLH-/HemoPh+, p-value<0.001 for HLH-/HemoPh- vs HLH+ and p-value<0.001 for HLH-/HemoPh+ vs HLH+.

**According to Bonferroni correction p-value=0.396 for HLH-/HemoPh- vs HLH-/HemoPh+, p-value=0.003 for HLH-/HemoPh- vs HLH+ and p-value=0.003 for HLH-/HemoPh+ vs HLH+.

***According to Bonferroni correction p-value=1.000 for HLH-/HemoPh- vs HLH-/HemoPh+, p-value<0.001 for HLH-/HemoPh- vs HLH+ and p-value=0.042 for HLH-/HemoPh+ vs HLH+.

****According to Bonferroni correction p-value=1.000 for HLH-/HemoPh- vs HLH/HemoPh+, p-value=0.018 for HLH-/HemoPh- vs HLH+ and p-value=0.429 for HLH-/HemoPh+ vs HLH+.

26

Table 7: Proposed criteria for HLH diagnosis in AML patients

AML patient undergoing intensive chemotherapy

and

Fever

Hepatosplenomegaly

Ferritin > 5000 µg/L

Prolonged neutropenia and/or thrombopenia (outside the expected range for chemotherapy-

induced myelosuppression, e.g, day 35)

Liver abnormalities

These criteria should lead to perform a bone marrow aspiration to reveal hemophagocytosis.

These proposed criteria should be improved by classical criteria for HLH such as genetic

defect of in genes of the cytotoxic pathway (PRF1, UNC13D, STXBP2, RAB27A, STX11,

SH2D1A, or XIAP), natural killer cell activity and soluble CD25.

27

Figure Legends

Figure 1: Distribution of anti-HLH treatments according to underlying infections

CS: corticosteroids (either prednisolone 1 mg/kg/d or dexamethasone 10 mg twice a day three

days then taper); IVIg: intravenous immunoglobulins (0.4 g/kg/d for 5 days or 1 g/kg/d for 2

days). Of note, 4 patients had concomitant viral and bacterial infection (1 was treated with

IVIg, 2 with CS + IVIg and 1 with CS + etoposide), 3 had viral and fungal infection treated

with IVIgand 3 other bacterial and fungal infection treated with IVIg.

Figure 2: Overall survival of HLH + and HLH- patients


Supplementary data

Methods

Endpoints

The primary endpoint of the study was overall survival. For each participant, the length of

follow-up corresponds to the period between the date of diagnosis and July 1, 2012 or the date

of death if the patient died during the study period. The response to treatment was evaluated

after full hematological recovery (e.g, when neutrophils and platelet counts were > 1 G/L and

> 100 G/L to document complete responses), or at day 35 in cases of prolonged aplasia, and

defined according to the international consensus criteria as complete response (CR) or

complete response with incomplete blood count recovery (CRi).15 Early death was defined as

death from any cause occurring between the start of chemotherapy and the response

assessment. Resistant disease, death in aplasia and relapses were defined according to Cheson

criteria.15 The duration of neutropenia was defined as the number of days between d1 of

chemotherapy and the first day with neutrophils higher than 0.5 G/L or death for those who

died with less than 0.5 G/L.

Statistical analysis

Statistical analysis was performed on STATA statistical software, release 11.2 (STATA

Corporation, College station, TX, USA). We described patients’ characteristics using number

and frequency for qualitative data and number, median and Inter-Quartile Range (IQR) for

quantitative data. Qualitative variables were compared between groups (HLH+, HLH-

/HemoPh+ and HLH-/HemoPh- patients) using the χ2-test (or Fisher’s exact test in the case of

small expected numbers). Student’s t-test was used to compare the distribution of quantitative

data (or Mann-Whitney’s test when distribution departed from normality or when

homoscedasticity was rejected). Differences in survival functions were tested using the Log-

Rank test. The independent impact of HLH on overall survival was assessed using a Cox

model adjusted for age, secondary AML, white blood cell count, cytogenetics, performance

status and consolidation treatment by autologous or allogeneic stem cell transplantation

(SCT). HLH+ patients first become at risk of HLH+ mortality at the date of HLH+ diagnosis

and patients treated with autologous or allogeneic SCT first become at risk of SCT mortality

at the date of SCT. Since the linearity hypothesis was not fully respected for WBC, this

continuous co-factor was transformed into ordered data: ≤ 50 G/L and > 50 G/L. The

proportional-hazard assumption was tested for each covariate by the “log-log” plot method

curves ((-ln{-ln(survival)}), for each category of nominal covariate, versus ln(analysis time)).

None of the assumptions could be rejected. Interactions between HLH and the independent

covariates were tested in the survival model (none were significant). All reported p-values

were two-sided and the significance threshold was < 0.05.

Supplementary figure.

Light microscopic representative images of May–Grünwald–Giemsa (MGG) stained bone marrow smear showing macrophagescontaining hematopoietic cells in their cytoplasm (x100). A, B, C. Erythroblast. D. Platelets.

A B

C D

hemophagocytic syndrome in acute myeloid leukemia patients

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